Name: isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes
Uploaded: 2018-05-31T13:00:00
Description: Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

The protocols below refer to the isolation of mouse MoDC, yet the overall principles may apply to other DC subsets cells, as well. 12 - 16-week-old C57Bl/6j mice were maintained in an American Association for the Accreditation of Laboratory Animal Care&#8211;accredited animal facility. All protocols were approved by Stanford University and Tel-Aviv University Institutional Animal Care and Use Committee.
1. Isolation of Tumor Associated Monocyte-Derived DC
<ol>
	<li>In a laminar flow hood, re...

<ol>
	<li>Steinman, R. M., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+dendritic+cells+into+medicine.">Taking dendritic cells into medicine.</a> Nature. 449 (7161), 419-426 (2007).</li><li>Palucka, K., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic-cell-based+therapeutic+cancer+vaccines.">Dendritic-cell-based therapeutic cancer vaccines.</a> Immunity. 39 (1), 38-48 (2013).</li><li>Mildner, A., Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+function+of+dendritic+cell+subsets.">Development and function of dendritic cell subsets.</a> Immunity. 40 (5), 642-656 (2014).</li><li>Merad, M., Sathe, P., Helft, J., Miller, J., Mortha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dendritic+cell+lineage:+ontogeny+and+function+of+dendritic+cells+and+their+subsets+in+the+steady+state+and+the+inflamed+setting.">The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.</a> Annual review of immunology. 31, 563-604 (2013).</li><li>Guilliams, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells,+monocytes+and+macrophages:+a+unified+nomenclature+based+on+ontogeny.">Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny.</a> Nature reviews. 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Given the large number of DC required for vaccinating mice (approximately 2-4 x 106 DC per one mouse), most of the vaccination strategies in mice are based on isolation of DC from BM and spleen followed by their ex vivo activation. However, attempts to activate tumor DC in vivo, using the same conditions for activating spleen and BM DC, have often been unsuccessful in producing effective immunity. In two subsequent publications, Carmi et al. have found that blood and tumor MoDC diffe...

Since their discovery, dendritic cells (DC) have been a focus of extensive research due to their unique ability to skew T cell differentiation1. Over the past several decades, an extensive research effort has sought to define the various DC subsets and their function during tumor progression and immunity 2. DCs are composed of heterogeneous cell populations that differ from each other in their pattern-recognition receptors, tissue distribution, and migratory and antigen presentation capabilities3,4,5. Compared to other DC subsets, monocyte-derived DC (MoDC) are far more abundant in tumors and can be easily generated from circulating or tumor-infiltrating monocytes6,7. Therefore, many clinical trials seeking to take advantage of their relative prevalence are based on in vivo and ex vivo manipulation of autologous MoDC in order to elicit T cell immunity 8,9.
Similarly, DC-based vaccination of experimental tumor models requires 2-3 serial injections, 5-7 days apart, of 1-2 x 106 activated DC pulsed with tumor antigens. Therefore, to achieve this large number of DC, most mouse studies have primarily used MoDC cultured from bone marrow (BM) precursors in GM-CSF for 7-9 days (IL-4 is not needed in the mouse setting)10,11. Nonetheless, given that GM-CSF knockout mice have overall normal DC compartment 12,13, and given the mixed populations obtained from that culture,14 the physiological relevance of these DC has been called into question.
Alternatively, DC may be routinely isolated from spleen cells. However, DC comprise only about 0.3-0.8% of total spleen cells (resulting in approximately 7 x 105 DC/spleen), and of these cells, only CD103+ DC and MoDC can migrate back to lymphoid organs. Since MoDCs comprise approximately 10-15% of splenic DC populations15,16, most isolation protocols yield approximately 1 x 105 MoDC per spleen. Expansion of MoDC can be achieved by injecting transfected B16 cells that secrete GM-CSF, resulting in a 100-fold increase in splenic MoDC17. However, the use of MoDC for developing DC vaccines is limited since this procedure cannot be done in humans and the obtained MoDC are already highly activated.
In addition to obtaining adequate numbers of DC, another challenge for developing effective DC vaccines against autologous cancer cells involves the lack of sufficient danger signals in the tumor setting to fully activate DC. Induction of co-stimulatory signals is usually achieved through activation of pattern-recognition receptors (PRR), or c-type lectin signaling pathways18,19,20,21. A further approach for activating DC exploits their ability to take up antigens through interactions with surface Fc&#947; receptors (Fc&#947;R). Indeed, a number of important manuscripts have shown that injection of MoDC from BM precursors activated with tumor-IgG IC can prevent tumor growth in prophylactic settings, and can lead to the eradication of established tumors22,23.
In two recent papers, Carmi et al. discovered that in contrast to BMDC and spleen DC, MoDC from the blood and tumors cannot respond to IgG IC without additional stimuli. This was found to be due to the presence of high intracellular levels of tyrosine phosphatases regulating Fc&#947;R signaling24,25. By defining a critical checkpoint in DC, this work provided an important insight into the requirements for successful DC-based vaccination. The requirement for additional stimuli to enable Fc&#947;R signaling, and presumably signaling from other lectin receptors utilizing a similar phosphorylation cascade, thus underscores the need for avoiding the priming of DC during their isolation.
Therefore, the present protocol describes the isolation of MoDC from blood and tumors, which differ markedly from BM and spleen DC, and highlights precautions worth considering during the process.

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE-Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>StemCell Technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19861</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C9697-50MG</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>PE-CD11c</td>
			<td>Biolegend</td>
			<td>117307</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD11b</td>
			<td>Biolegend</td>
			<td>101211</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 MHCII</td>
			<td>Biolegend</td>
			<td>107641</td>
			<td></td>
		</tr>
		<tr>
			<td>AF48- CD86</td>
			<td>Biolegend</td>
			<td>105017</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cy7-Ly-C6</td>
			<td>Biolegend</td>
			<td>108423</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7-CD15</td>
			<td>Biolegend</td>
			<td>135523</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

We initially compared the capacity of antibodies from na&#239;ve syngeneic and allogeneic mice to bind to tumor cells. To this end, B16F10 and LMP tumor cell lines were fixed in paraformaldehyde and washed extensively. B16F10 is a melanoma cell line, which was originally isolated from lung metastases in C57Bl/6 mice. LMP is a pancreatic tumor cell that was isolated from KrasG12D/+, LSL-Trp53R172H/+, and Pdx-1-Cre mice, and grows steadily in 129F1 mice. To obtain IC, tumor cells were incub...

Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

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Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...